Most of the major laboratories working on the Tat vaccine have failed to pay attention to basics. (1) They over looked the fact that to control viral infection, Tat must be presented to the immune system as a DNA vaccine, not as a protein or toxoid. (2) These workers also underestimated the importance of immune-modulation of Tat to make it immunodominant. (3) Importantly, some workers disregarded the need to modulate Tat to reduce or alleviate its toxic properties and used native Tat in their vaccines. While others realized the importance of attenuating Tat but adapted an erroneous strategy of chemically modifying Tat to convert it into less toxic form of a Tat toxoid. Tat toxoid is known to differ from the native Tat in the quality of immune responses it would induce in immunizations.
HIV Vaccine Design Traditionally been Dominated by Structural Proteins Like Env:
HIV vaccine development traditionally depended on structural proteins like env, gag and regulatory protein Nef all of which are immunodominant. Antibody response to env is certainly critical to prevent or reduce the rate of infection at the entry level. Recent studies have demonstrated the importance of cell-mediated immune responses to gag in restricting viral proliferation in vivo (Kiepiela et al., 2007; Novitsky et al., 2003; Novitsky et al., 2006). In contrast, evidence is also available that gag vaccines failed to induce protective immune response (Saini et al., 2007; Putkonen et al., 1998). In a head-to-head comparison of Tat vs gag immune responses in a primate model, immune responses to Tat but not gag provided protection against viral challenge (Stittelaar et al., 2002). Inclusion of an antigen like Nef in vaccine design could be risky given that Nef is an accessory protein (not an essential protein unlike env, gag or Tat), the presence of which is not critical for the survival of the virus and the virus could efficiently develop resistance against Nef. Although env vaccines conferred protection against autologous viral strains, antigenic variation is a challenge for vaccine design (Osmanov et al., 1996). Further, most of the clinical trials using env and other structural antigens did not provide protective efficacy (Veljkovic et al., 2003; Kaiser, 2008; Bubnoff, 2007; Steinbrook, 2007).
A Need for Multi-Component HIV Vaccine:
In this backdrop, inclusion multiple antigens in HIV vaccine design and optimizing each individual antigen for efficient immune response is essential. A need for developing multi-component vaccines is being increasingly realized, to induce broader immune responses against the viral infection, by incorporating multiple viral antigens (Ho and Huang, 2002). Extensive work from various laboratories has identified the viral structural proteins, gag and pol, and viral regulatory proteins Nef, Tat and Rev, as potential candidates for vaccine development (Calarota et al., 1999; Evans et al., 1999; Putkonen et al., 1998). Of these non-env candidates, Tat occupies a special place for several reasons.
Significance of Tat for HIV Vaccine Development:
First of all, the functional importance of this viral antigen to the infectivity of the virus (Gallo, 1999; Jeang et al., 1999; Rubartelli et al., 1998; Rusnati and Presta, 2002), and the existence of an inverse correlation between immune responses to Tat and disease progression (Allen et al., 2000; Re et al., 1995; Re et al., 2001; Reiss et al., 1990; Zagury et al., 1998b; van Baalen et al., 1997) make Tat an important candidate vaccine. Several studies showed that immune responses to Tat, humoral or cellular, appear to have protected against disease progression or viral load (Richardson et al., 2003; Re et al., 2001; Zagury et al., 1998b; van Baalen et al., 1997; Re et al., 1996; Rodman et al., 1992; Reiss et al., 1990; Wieland et al., 1990) although a few studies demonstrated absence of such effect (Senkaali et al., 2008). Tat is expressed early in the viral life cycle and is functionally important for its infectivity and pathogenicity (Jeang et al., 1999). In addition to regulating viral gene expression, Tat modulates expression of various genes of the host. Further, Tat is secreted extracellularly and the extracellular Tat governs viral latency and contributes to disease progression (Noonan and Albini, 2000). Inducing cellular as well as humoral immune responses against Tat is critical owing to its early expression in the viral life cycle and to its extracellular secretion (Goldstein, 1996; Rusnati and Presta, 2002). Lastly, as a consequence of its pleiotropic biologic functions, a variety of functional assays are available for Tat, to study the inhibitory effect of immune components on its biological functions.
The Cysteine-Rich Domain and Basic Domains of Tat Regulate Important Biological Functions of Tat:
The Tat protein of HIV-1 is a small polypeptide of 101 amino acid residues encoded by two exons. Like many transcription factors, Tat is structurally flexible (Dyson and Wright, 2005) and as a result, its crystal structure could not be determined by X-ray crystallography. Structural prediction of Tat by NMR spectroscopy suggests lack of obvious secondary structures in Tat (Peloponese, Jr. et al., 2000; Gregoire et al., 2001; Shojania and O'neil, 2006). Depending on the nature of amino acid distribution, five conserved functional domains have been identified in Tat exon-1 (Jeang et al., 1999). These include (1) the proline-rich N-terminal region consisting of residues 1-21 is predicted to assume an α-helical structure, (2) the cysteine-rich domain (CRD) consisting of residues 22-37 and makes an intra-molecular disulphide bond, (3) the core domain consisting of the residues 38-48 makes the third domain, (4) the basic domain consists of the residues 49-57 and (5) the C-terminal region consisting of the residues 58-72 is rich in glutamine. The CRD and the core domain together constitute the activation domain that regulates viral promoter transactivation (Jeang et al., 1999). In addition, CRD regulates many more functions including lymphocyte chemotaxis (Albini et al., 1998) and triggering cellular apoptosis (Mishra et al., 2007). The basic domain (BD), rich in arginines regulates several important biological functions of Tat. These functions include nuclear localization of Tat, crossing membranes while entering or exiting the cell, binding to the uridine-rich bulge motif in the HIV TAR mRNA, and for dimerization of Tat.
Optimization of Vaccine Performance by Incorporating Diverse Molecular Strategies:
A wide range of molecular strategies have been employed to enhance performance of different types of vaccines. The strategies encompass an indeed wide array of strategies to improve protein expression, transcript stabilization, antigen processing and presentation, antigen delivery, coadministration of immune modulatory factors, recruiting innate immune components and many more. Reviewing all these components is beyond the scope of this section. Engineering the pan antigen DR epitope (PADRE), a universal HLA DR binding peptide (Alexander et al., 1994b), or other T-helper epitopes into antigens is one of the molecular strategies extensively used by many groups to enhance antigen-specific immune responses (Alexander et al., 1998). Given that peptide antigens are less immunogenic, PADRE epitope has been widely used to enhance immunogenicity of this form of vaccines (Beebe et al., 2007; Decroix et al., 2002; Fitzmaurice et al., 1996; Hsu et al., 1999; Olszewska et al., 2000), including that of HIV-1 env (Belyakov et al., 1998). Use of T-helper epitope into protein vaccines is less common although some examples are available (Greenstein et al., 1992; Rosa et al., 2004). Carbohydrate vaccines, derived from pathogenic organisms, that are least immunogenic intrinsically too shown to become immunogenic after conjugating such substrates to PADRE epitope (Alexander et al., 2004; Belot et al., 2005). Use of PADRE for mucosal vaccines has been documented (Decroix et al., 2002; Belyakov et al., 1998). A large quantum of effort has been directed against diverse type of cancers by generating cancer-specific peptides or antigens that are molecularly linked to T-helper epitopes (Beebe et al., 2007; Mansour et al., 2007; Stevenson et al., 2004b; van Bergen et al., 2000). T-helper epitopes have been engineered into DNA vaccines to augment their performance against viral infections (Hsu et al., 1999; Gao et al., 2004; Hung et al., 2007; Kim et al., 2007), including HIV-1 (Gorse et al., 2008; Newman et al., 2002). Polyclonal antisera with high antibody titers were raised in experimental animals against more than a hundred different antigens when these antigens were expressed as chimeras of PADRE epitope suggesting generic and wide application of T-help recruitment to a broad range of antigens (Chambers and Johnston, 2003). Recruitment of T-help through PADRE T-helper epitope has also been documented against parasite infections (Rosa et al., 2004), and even auto-immune disorders like Alzheimer's (Agadjanyan et al., 2005) or experimental autoimmune encephalitis (Uyttenhove et al., 2004).
Limitations of the Existing Tat Vaccines:
Despite all its merits, initial attempts of Tat vaccine met with limited success to the extent that there were doubts as per the rationale of Tat as a candidate vaccine. The primary reason why Tat vaccine did not yield expected results is because all the previous strategies ignored the basics while designing vaccines. Several technical challenges must be addressed before expecting Tat to function as a preventive or therapeutic vaccine. Some of the important limitations of the Tat vaccines can be broadly classified into three categories which have been described briefly below (a) poor immune response to Tat, (b) safety concerns since Tat is a toxin and an immunomodulator and (c) restricted antigen presentation as a protein.
(a) Tat is Non-Immunodominant:
Tat is a small nuclear protein that lacks potential T-helper epitopes. Although T-helper epitopes have been mapped in Tat (Blazevic et al., 1993; Ramakrishna et al., 2004; Ranki et al., 1997; Silvera et al., 2002), in natural infection, several lines of evidence suggest that these T-helper epitopes may not be strong enough. Only a fraction, 10-15% (data from JNCASR laboratory), of the seropositive subjects make anti-Tat humoral immune response (Krone et al., 1988; Reiss et al., 1990; Wieland et al., 1990). Of these subjects, only a minority show isotope switching to IgG indicating lack of efficient T-help (Venkatesh P K et al, manuscript in preparation). Likewise, cell-mediated immune responses to Tat were also shown to be scarce in natural infection (Borrow et al., 1994; Goulder et al., 2001; Lieberman et al., 1997; Masemola et al., 2004; Lamhamedi-Chemadi et al., 1992). The non-immunodominant nature of Tat must be an intrinsic property of Tat given that in experimental immunization too strong immune responses are not seen in primate (Putkonen et al., 1998; Belliard et al., 2005; Pauza et al., 2000) or human (Calarota et al., 1999; Hejdeman et al., 2004) studies. Non-availability of sufficient quantity of Tat in extra-cellular milieu could also be a contributory factor for non-immunodominant nature of Tat in natural infection. Although Tat is believed to be secreted extracellularly (eTat), the data in support of this hypothesis are scanty and wanting.
The foregoing suggests that molecular strategies are required to enhance immune responses induced by Tat for this antigen ever to become a candidate vaccine. Nearly all the previous attempts ignored this critical issue and used Tat as a protein, toxoid or DNA without means to enhance immune response.
(b) Tat being a Toxin Raises Safety Concerns:
As an extracellular viral factor, eTat is believed to possess pleiotropic effects on host cells and host immune system to enhance viral pathogenesis and infectivity. Some of these properties of eTat could have serious consequences especially in immune-compromised subjects (Huigen et al., 2004).                i. Latent virus activation: eTat could activate latent viruses thus contributing to spreading of the viral infection.        ii. Apoptosis of the lymphocytes: eTat could program uninfected T-lymphocytes (Li et al., 1995), B-cells (Huang et al., 1997) and monocytes, to commit to apoptosis thus increasing the chances immune-suppression in HIV infected subjects. Tat can also inhibit NK cell function contributing to NK cell dysfunction (Zocchi et al., 1998)        iii. Coreceptor upregulation: Tat can upregulate expression of coreceptors CCR5 and CXCR4 on target T-cells thus increasing the chances of viral infection (Huang et al., 1998). Likewise, Tat can modulate expression of a broad range of host genes with serious consequences for the host (Giacca, 2004).        iv. Neuropathogenesis: Direct exposure of neurons and astrocytes to Tat is known to enhance cell death leading to neurologic consequences including enhanced dementia (Nath et al., 1998; Mishra et al., 2007).        v. Perturbing cytokine homeostasis: Tat can induce cells of diverse phenotype to secrete cytokines and/or chemokines thereby actively perturbing the cytokine homeostasis in the body and consequently contributing to overall immune-suppression (Lafrenie et al., 1997; Nath et al., 1999).        vi. Immunosuppression: Tat activates TNF-α secretion from macrophages leading to immune-suppression (Zagury et al., 1998a) or through TGF-β (Reinhold et al., 1999). Tat could directly inhibit T-cell proliferation (Zagury et al., 1998a; Viscidi et al., 1989). Coexpression of Tat inhibited immune responses to env through the mediation of IL-10 activation (Gupta et al., 2008). In contrast, coexpression of Tat was shown to broaden immune recognition of HIV-1 gag and env demonstrating adjuvant properties (Gavioli et al., 2008). Although Tat is also known to be an immunoactivator (Fanales-Belasio et al., 2002; Gavioli et al., 2004), the conditions that regulate the fine balance between these contradictory functions of Tat are not well understood.Tat Vaccine Controversy:        
The recent controversy around the Tat vaccine developed by Dr. Barbara Ensoli's group in Italy revolves essentially around these safety concerns of Tat (Cohen, 2007) and Controversy Over European Framework Programme AIDS Vaccines (ISIS Press Release Dec. 10, 2007) (http://www.isis.org.uk/ControversyAIDSvaccines.php). The vaccine developed by this group consists of the functional Tat protein that could have potential hazards associated for human use (Ensoli et al., 2006). No strategies have been employed to answer the question of safety of this Tat vaccine candidate.
Tat Toxoid and Other Inactive Forms of Tat:
Attempts have been made to formulate Tat protein as a toxoid by chemical treatment (Gringeri et al., 1998; Le Buanec and Bizzini, 2000). Tat toxoid was shown to be safe and also generated moderate immune responses in humans (Gringeri et al., 1998; Gringeri et al., 1999; Noonan et al., 2003; Moreau et al., 2004) and in primates (Pauza et al., 2000; Richardson et al., 2002; Silvera et al., 2002). Although several studies demonstrated immunogenicity of Tat taxoid, often comparable to the Tat protein, evidence also exists that Tat toxoid may generate qualitatively different immune response as compared to the native antigen (Tikhonov et al., 2003; Yang et al., 2003). However, native, but not oxidized, Tat promoted maturation of monocyte-derived dendritic cells and efficient antigen presentation from them suggesting that functional Tat could be a superior vaccine candidate than the attenuated forms (Fanales-Belasio et al., 2002). Additionally, native Tat protein also modulated the subunit composition of the immunoprotasomes leading to augmented antigen processing (Gavioli et al., 2004; Remoli et al., 2006). Tat mutants inactive for transactivation have been tested in mice but no progress reported beyond this animal model (Caselli et al., 1999; Mayol et al., 2007). Oxidized Tat was proposed to be a safe format for vaccination (Cohen et al., 1999).
(c) Tat as a Protein or Toxoid May not Access the MHC Class-I Compartment Efficiently:
Tat predominantly is an intra-cellular protein although experimental evidence suggests its secretion into the body fluids (Chang et al., 1997). Further, Tat is not exposed on the surface of the virus. Cell-mediate immune responses to Tat, therefore, should be the predominant component to restrict viral expansion in vivo although antibodies do play a significant role. Majority of the previous strategies used Tat as a recombinant protein or toxoid in primate immunization studies (Cafaro et al., 1999; Ensoli and Cafaro, 2000; Pauza et al., 2000; Richardson et al., 2002; Silvera et al., 2002; Tikhonov et al., 2003) or human clinical trial (Ensoli et al., 2006). As proteins, these antigens are less likely to access the MHC-I compartment to stimulate efficient anti-viral cell-mediated immune response. Although Tat protein is known to be cross-presented to MHC-I compartment (Kim et al., 1997), it not likely to be a predominant pathway of antigen presentation. The absence of strong cellular immune responses in the previously reported studies underlies the importance of targeting Tat to MHC-I compartment for vaccine development. Recombinant viruses efficiently introduce encoded antigens into MHC-I pathway, immune intervention, however, could interfere with immune responses (de et al., 2008; Willis et al., 2006). Further, preexisting immune response to the viral vector is a significant problem that limits recombinant vector-mediated antigen delivery (Bangari and Mittal, 2006). DNA vaccine, therefore, is an ideal medium for antigen delivery given that this form of vaccination can stimulate strong immune responses akin to viral vectors. DNA vaccines, however, have several technical challenges that must be addressed before they could be used as a reliable medium of immunization (Dean et al., 2005).
This project proposal enlists several potentially important molecular and immunologic features to address several critical challenges of the Tat vaccine as discussed above.